Non-caloric sweetener

ABSTRACT

Disclosed is a steviol glycoside referred to as rebaudioside D2. Rebaudioside D2 has five β-D-glucosyl units connected to the aglycone steviol. Also disclosed are methods for producing rebaudioside D2, a UDP-glycosyltransferase fusion enzyme, and methods for producing rebaudioside D and rebaudioside E.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. application Ser. No.15/016,750, entitled “NON-CALORIC SWEETENER” filed on Feb. 5, 2016,which is a divisional of U.S. application Ser. No. 14/269,435, entitled“NON-CALORIC SWEETENER” filed on May 5, 2014. The entire contents ofthese applications are incorporated herein by reference in theirentirety.

STATEMENT IN SUPPORT FOR FILING A SEQUENCE LISTING

A computer readable form of the Sequence Listing containing the filenamed “C149770008U505-SEQ-AM.txt”, which is 32,681 bytes in size (asmeasured in MICROSOFT WINDOWS® EXPLORER), is provided herewith and isherein incorporated by reference. This Sequence Listing consists of SEQID NOs:1-6.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to natural sweeteners. Moreparticularly, the present disclosure relates to a non-caloric sweetenerand methods for synthesizing the non-caloric sweetener.

Steviol glycosides are natural products isolated from Stevia rebaudianaleaves. Steviol glycosides are widely used as high intensity,low-calorie sweeteners and are significantly sweeter than sucrose.Naturally occurring steviol glycosides share the same basic steviolstructure, but differ in the content of carbohydrate residues (e.g.,glucose, rhamnose and xylose residues) at the C13 and C19 positions.Steviol glycosides with known structures include, steviol, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F and dulcoside A (see e.g., Table 1).

TABLE 1 Steviol glycosides. Molecular Molecular Name Structure FormulaWeight Steviol

C₂₀H₃₀O₃ 318 Stevioside

C₃₈H₆₀O₁₈ 804 Rebaudioside A

C₄₄H₇₀O₂₃ 966 Rebaudioside- B

C₃₈H₆₀O₁₈ 804 Rebaudioside C

C₄₄H₇₀O₂₂ 950 Rebaudioside D

C₅₀H₈₀O₂₈ 1128 Rebaudioside E

C₄₄H₇₀O₂₃ 966 Rebaudioside F

C₄₃H₆₈O₂₂ 936 Rebaudioside D2

C₅₀H₈₀O₂₈ 1128 Dulcoside A

C₃₈H₆₀O₁₇ 788

On a dry weight basis, stevioside, rebaudioside A, rebaudioside C, anddulcoside A, account for 9.1, 3.8, 0.6, and 0.3% of the total weight ofthe steviol glycosides in the leaves, respectively, while the othersteviol glucosides are present in much lower amounts. Extracts from theStevia rebaudiana plant are commercially available, which typicallycontain stevioside and rebaudioside A as primary compounds. The othersteviol glycosides typically are present in the stevia extract as minorcomponents. For example, the amount of rebaudioside A in commercialpreparations can vary from about 20% to more than 90% of the totalsteviol glycoside content, while the amount of rebaudioside B can beabout 1-2%, the amount of rebaudioside C can be about 7-15%, and theamount of rebaudioside D can be about 2% of the total steviolglycosides.

Steviol glycosides differ from each other not only by molecularstructure, but also by their taste properties. For example, differentsteviol glycosides have different degrees of sweetness and after-taste.Stevioside, for example, is 100-150 times sweeter than sucrose, but hasa bitter after-taste. Rebaudioside A and rebaudioside E, for example,are 250-450 times sweeter than sucrose and have less of an after-tastethan stevioside. Rebaudioside C is between 40-60 times sweeter thansucrose. Dulcoside A is about 30 times sweeter than sucrose.

The majority of steviol glycosides are formed by several glycosylationreactions of steviol, which are typically catalyzed by theUDP-glycosyltransferases (UGTs) using uridine 5′-diphosphoglucose(UDP-glucose) as a donor of the sugar moiety. UGTs in plants make up avery diverse group of enzymes that transfer a glucose residue fromUDP-glucose to steviol. For example, glycosylation of the C-3′ of theC-13-O-glucose of stevioside yields rebaudioside A; and glycosylation ofthe C-2′ of the 19-O-glucose of the stevioside yields rebaudioside E.Further glycosylation of rebaudioside A (at C-19-O-glucose) orrebaudioside E (at C-13-O-glucose) produces rebaudioside D. (FIG. 1).

Alternative sweeteners are receiving increasing attention due toawareness of many diseases in conjunction with the consumption ofhigh-sugar foods and beverages. Although artificial sweeteners areavailable, many artificial sweeteners such as dulcin, sodium cyclamateand saccharin have been banned or restricted by some countries due toconcerns over their safety. Therefore, non-caloric sweeteners of naturalorigin are becoming increasingly popular. One of the main obstacles forthe widespread use of stevia sweeteners are their undesirable tasteattributes. Accordingly, there exists a need to develop alternativesweeteners and methods for their production to provide the bestcombination of sweetness potency and flavor profile.

SUMMARY OF THE DISCLOSURE

The present disclosure relates generally to natural sweeteners. Moreparticularly, the present disclosure relates to a non-caloric sweetenerand methods for synthesizing the non-caloric sweetener. The presentdisclosure also relates to a enzyme that can be used to prepare thenon-caloric sweetener.

Steviol Glycoside—Synthetic Rebaudioside D2.

In one aspect, the present disclosure is directed to a syntheticrebaudioside (rebaudioside D2) consisting of a chemical structure:

Method of Producing Rebaudioside D2 from Rebaudioside E.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D2 from rebaudioside E. The method comprisespreparing a reaction mixture comprising rebaudioside E; a substrateselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); and a uridine diposphoglycosyltransferase (UDP-glycosyltransferase) selected from the groupconsisting of a uridine diphospho glycosyltransferase and a UDP-glycosyltransferase fusion enzyme comprising a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain; andincubating the reaction mixture for a sufficient time to producerebaudioside D2, wherein a glucose is covalently coupled to therebaudioside E to produce rebaudioside D2.

Method of Producing Rebaudioside E and Rebaudioside D2 from Stevioside.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside E and rebaudioside D2 from stevioside. Themethod comprises preparing a reaction mixture comprising stevioside; asubstrate selected from the group consisting of sucrose, uridinediphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and auridine dipospho glycosyltransferase (UDP-glycosyltransferase) selectedfrom the group consisting of a uridine diphospho glycosyltransferase anda UDP-glycosyltransferase fusion enzyme comprising a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain; andincubating the reaction mixture for a sufficient time to producerebaudioside E and rebaudioside D2, wherein a glucose is covalentlycoupled to the stevioside to produce a rebaudioside E intermediate andwherein a glucose is covalently coupled to the rebaudioside Eintermediate to produce rebaudioside D2.

UDP-Glycosyltransferase Fusion Enzyme (“EUS”).

In another aspect, the present disclosure is directed to aUDP-glycosyltransferase fusion enzyme (referred to herein as “EUS”). TheUDP-glycosyltransferase fusion enzyme comprises a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain. TheUDP-glycosyltransferase fusion enzyme demonstrates 1,2-β glycosidiclinkage and 1,6-β glycosidic linkage enzymatic activities as well assucrose synthase activity.

Method for Producing Rebaudioside D from Rebaudioside A.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D from rebaudioside A. The method comprisespreparing a reaction mixture comprising rebaudioside A; a substrateselected from the group consisting of sucrose, uridine diphosphate (UDP)and uridine diphosphate-glucose (UDP-glucose); and aUDP-glycosyltransferase selected from the group consisting of a uridinediphospho glycosyltransferase and a UDP-glycosyltransferase fusionenzyme (EUS) comprising a uridine diphospho glycosyltransferase domaincoupled to a sucrose synthase domain; and incubating the reactionmixture for a sufficient time to produce rebaudioside D, wherein aglucose is covalently coupled to the rebaudioside A to producerebaudioside D.

In another aspect, the present disclosure is directed to an orallyconsumable product comprising a sweetening amount of rebaudioside D2selected from the group consisting of a beverage product and aconsumable product.

In another aspect, the present disclosure is directed to a beverageproduct comprising a sweetening amount of rebaudioside D2. Therebaudioside D2 is present in the beverage product at a concentration ofabout 5 ppm to about 100 ppm. In some embodiments, low concentrations ofrebaudioside D2, e.g., below 100 ppm, has an equivalent sweetness tosucrose solutions having concentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure is directed to a consumableproduct comprising a sweetening amount of rebaudioside D2. Therebaudioside D2 is present in the consumable product at a concentrationof about 5 ppm to about 100 ppm. In some embodiments, low concentrationsof rebaudioside D2, e.g., below 100 ppm, has an equivalent sweetness tosucrose solutions having concentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

In certain embodiments that can be combined with any of the precedingembodiments, the rebaudioside D2 the only sweetener, and the product hasa sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrosesolution. In certain embodiments that may be combined with any of thepreceding embodiments, the orally consumable product further includes anadditional sweetener, where the product has a sweetness intensityequivalent to about 1% to about 10% (w/v-%) sucrose solution. In certainembodiments that may be combined with any of the preceding embodiments,every sweetening ingredient in the product is a high intensitysweetener. In certain embodiments that may be combined with any of thepreceding embodiments, every sweetening ingredient in the product is anatural high intensity sweetener. In certain embodiments that may becombined with any of the preceding embodiments, the additional sweetenercontains one or more sweeteners selected from a stevia extract, asteviol glycoside, stevioside, rebaudioside A, rebaudioside B,rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,dulcoside A, rubusoside, steviolbioside, sucrose, high fructose cornsyrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol,xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame,sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidindihydrochalcone (NDHC), rubusoside, mogroside IV, siamenoside I,mogroside V, monatin, thaumatin, monellin, brazzein, L-alanine, glycine,Lo Han Guo, hernandulcin, phyllodulcin, trilobtain, and combinationsthereof. In certain embodiments that may be combined with any of thepreceding embodiments, the beverage product and consumable productfurther includes one or more additives selected from a carbohydrate, apolyol, an amino acid or salt thereof, a poly-amino acid or saltthereof, a sugar acid or salt thereof, a nucleotide, an organic acid, aninorganic acid, an organic salt, an organic acid salt, an organic basesalt, an inorganic salt, a bitter compound, a flavorant, a flavoringingredient, an astringent compound, a protein, a protein hydrolysate, asurfactant, an emulsifier, a flavonoids, an alcohol, a polymer, andcombinations thereof. In certain embodiments that may be combined withany of the preceding embodiments, the rebaudioside D2 has a purity ofabout 50% to about 100% by weight before it is added into the product.In certain embodiments that may be combined with any of the precedingembodiments, the rebaudioside D2 in the product is a rebaudioside D2polymorph or amorphous rebaudioside D2. In certain embodiments that maybe combined with any of the preceding embodiments, the rebaudioside D2in the product is a rebaudioside D2 stereoisomer.

Other aspects of the present disclosure relate to a method of preparinga beverage product and a consumable product by including purifiedrebaudioside D2 into the product or into the ingredients for making thebeverage product and the consumable product, where rebaudioside D2 ispresent in the product at a concentration of from about 5 ppm to about100 ppm. Other aspects of the present disclosure relate to a method forenhancing the sweetness of a beverage product and a consumable productby adding from about 5 ppm to about 100 ppm of purified rebaudioside D2into the beverage product and the consumable product, where the addedrebaudioside D2 enhances the sweetness of the beverage product and theconsumable product, as compared to a corresponding a beverage productand a consumable product lacking the purified rebaudioside D2.

In certain embodiments that may be combined with any of the precedingembodiments, the rebaudioside D2 is the only sweetener, and the producthas a sweetness intensity equivalent to about 1% to about 4% (w/v-%)sucrose solution. In certain embodiments that may be combined with anyof the preceding embodiments, the method further includes adding anadditional sweetener, where the product has a sweetness intensityequivalent to about 1% to about 10% (w/v-%) sucrose solution.

Other aspects of the present disclosure relate to a method for preparinga sweetened beverage product or a sweetened consumable product by: a)providing a beverage product or a consumable product containing one ormore sweetener; and b) adding from about 5 ppm to about 100 ppm ofpurified rebaudioside D2 into the beverage product or the consumableproduct.

In certain embodiments that may be combined with any of the precedingembodiments, the method further includes adding one or more additives tothe beverage product or the consumable product. In certain embodimentsthat may be combined with any of the preceding embodiments, the orallyconsumable product further contains one or more additives. In certainembodiments that may be combined with any of the preceding embodiments,the one or more additives are selected from a carbohydrate, a polyol, anamino acid or salt thereof, a poly-amino acid or salt thereof, a sugaracid or salt thereof, a nucleotide, an organic acid, an inorganic acid,an organic salt, an organic acid salt, an organic base salt, aninorganic salt, a bitter compound, a flavorant, a flavoring ingredient,an astringent compound, a protein, a protein hydrolysate, a surfactant,an emulsifier, a flavonoids, an alcohol, a polymer, and combinationsthereof. In certain embodiments that may be combined with any of thepreceding embodiments, every sweetening ingredient in the product is ahigh intensity sweetener. In certain embodiments that may be combinedwith any of the preceding embodiments, every sweetening ingredient inthe product is a natural high intensity sweetener. In certainembodiments that may be combined with any of the preceding embodiments,the sweetener is selected from a stevia extract, a steviol glycoside,stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudiosideD, rebaudioside E, rebaudioside F, dulcoside A, rubusoside,steviolbioside, sucrose, high fructose corn syrup, fructose, glucose,xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol,inositol, AceK, aspartame, neotame, sucralose, saccharine, naringindihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC),rubusoside, mogroside IV, siamenoside I, mogroside V, monatin,thaumatin, monellin, brazzein, L-alanine, glycine, Lo Han Guo,hernandulcin, phyllodulcin, trilobtain, and combinations thereof. Incertain embodiments that may be combined with any of the precedingembodiments, the rebaudioside D2 has a purity of about 50% to about 100%by weight before it is added into the product. In certain embodimentsthat may be combined with any of the preceding embodiments, therebaudioside D2 in the product is a rebaudioside D2 polymorph oramorphous rebaudioside D2.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIGS. 1A-1C are schematics illustrating the pathways of steviolglycoside biosynthesis from stevioside.

FIGS. 2A and 2B show the SDS-PAGE analysis of the purified recombinantUDP-glycosyltransferase enzyme (EUGT11) and the purified recombinantUDP-glycosyltransferase fusion enzyme (EUS), as discussed in Example 1.

FIGS. 3A-3G are graphs showing the HPLC retention times of stevioside(“Ste”), rebaudioside A (“Reb A”) and rebaudioside D (“Reb D”) standards(FIG. 3A); rebaudioside D enzymatically produced by EUS at 14 hours(FIG. 3B); rebaudioside D enzymatically produced by EUGT11 at 14 hours(FIG. 3C); rebaudioside D enzymatically produced by the UGT-SUS(EUGT11-AtSUS1) coupling system at 14 hours (FIG. 3D); rebaudioside Denzymatically produced by EUS at 24 hours (FIG. 3E); rebaudioside Denzymatically produced by EUGT11 at 24 hours (FIG. 3F); and rebaudiosideD enzymatically produced by the UGT-SUS (EUGT11-AtSUS1) coupling systemat 24 hours (FIG. 3G), as discussed in Example 2.

FIGS. 4A-4G are graphs showing the HPLC retention times of stevioside(“Ste”), rebaudioside A (“Reb A”) and rebaudioside D (“Reb D”) standards(FIG. 4A); rebaudioside D2 (“Reb D2”) enzymatically produced by EUS at14 hours (FIG. 4B); rebaudioside E (“Reb E”) enzymatically produced byEUGT11 at 14 hours (FIG. 4C); rebaudioside D2 enzymatically produced bythe UGT-SUS (EUGT11-AtSUS1) coupling system at 14 hours (FIG. 4D);rebaudioside D2 (“Reb D2”) enzymatically produced by EUS at 24 hours(FIG. 4E); rebaudioside E (“Reb E”) enzymatically produced by EUGT11 at24 hours (FIG. 4F); and rebaudioside D2 enzymatically produced by theUGT-SUS (EUGT11-AtSUS1) coupling system at 24 hours (FIG. 4G), asdiscussed in Example 3.

FIGS. 5A-5J are graphs showing the HPLC retention times of rebaudiosideD (“Reb-D”) standard (FIG. 5A); rebaudioside E (“Reb-E”) standard (FIG.5B); rebaudioside D2 (“Reb D2”) enzymatically produced by EUGT11 at 12hours (FIG. 5C); the UGT-SUS (EUGT11-SUS1) coupling system at 12 hours(FIG. 5D) and EUS at 12 hours (FIG. 5E); rebaudioside D (“Reb D”)enzymatically produced by a UGT76G1-AtSUS1 coupling system at 12 hours(FIG. 5F); rebaudioside D2 enzymatically produced by EUGT11 at 24 hours(FIG. 5G); rebaudioside D2 enzymatically produced by UGT-SUS(EUGT11-SUS1) coupling system at 24 hours (FIG. 5H); rebaudioside D2enzymatically produced by EUS at 24 hours (FIG. 5I); and rebaudioside Denzymatically produced by a UGT76G1-AtSUS1 coupling system at 24 hours(FIG. 5J), as discussed in Example 4.

FIGS. 6A-6B show the chemical structures of rebaudioside D2 andrebaudioside E, as discussed in Example 5.

FIG. 7 is a chemical structure of rebaudioside D2 illustrating the keyTOCSY and HMBC correlations, as discussed in Example 5.

FIGS. 8A-8C show the chemical structures of rebaudioside D2,rebaudioside E and rebaudioside D, as discussed in Example 5.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or testing of the present disclosure, the preferredmaterials and methods are described below.

The term “complementary” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and isused without limitation to describe the relationship between nucleotidebases that are capable to hybridizing to one another. For example, withrespect to DNA, adenosine is complementary to thymine and cytosine iscomplementary to guanine. Accordingly, the subject technology alsoincludes isolated nucleic acid fragments that are complementary to thecomplete sequences as reported in the accompanying Sequence Listing aswell as those substantially similar nucleic acid sequences.

The terms “nucleic acid” and “nucleotide” are used according to theirrespective ordinary and customary meanings as understood by a person ofordinary skill in the art, and are used without limitation to refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar tonaturally-occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified or degenerate variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated.

The term “isolated” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and whenused in the context of an isolated nucleic acid or an isolatedpolypeptide, is used without limitation to refer to a nucleic acid orpolypeptide that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form or can exist ina non-native environment such as, for example, in a transgenic hostcell.

The terms “incubating” and “incubation” as used herein refers to aprocess of mixing two or more chemical or biological entities (such as achemical compound and an enzyme) and allowing them to interact underconditions favorable for producing a steviol glycoside composition.

The term “degenerate variant” refers to a nucleic acid sequence having aresidue sequence that differs from a reference nucleic acid sequence byone or more degenerate codon substitutions. Degenerate codonsubstitutions can be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed base and/or deoxyinosine residues. A nucleic acid sequence and allof its degenerate variants will express the same amino acid orpolypeptide.

The terms “polypeptide,” “protein,” and “peptide” are used according totheir respective ordinary and customary meanings as understood by aperson of ordinary skill in the art; the three terms are sometimes usedinterchangeably, and are used without limitation to refer to a polymerof amino acids, or amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “polypeptide” as used herein refers to peptides, polypeptides, andproteins, unless otherwise noted. The terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein when referring to apolynucleotide product. Thus, exemplary polypeptides includepolynucleotide products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants, andanalogs of the foregoing.

The terms “polypeptide fragment” and “fragment,” when used in referenceto a reference polypeptide, are used according to their ordinary andcustomary meanings to a person of ordinary skill in the art, and areused without limitation to refer to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both.

The term “functional fragment” of a polypeptide or protein refers to apeptide fragment that is a portion of the full length polypeptide orprotein, and has substantially the same biological activity, or carriesout substantially the same function as the full length polypeptide orprotein (e.g., carrying out the same enzymatic reaction).

The terms “variant polypeptide,” “modified amino acid sequence” or“modified polypeptide,” which are used interchangeably, refer to anamino acid sequence that is different from the reference polypeptide byone or more amino acids, e.g., by one or more amino acid substitutions,deletions, and/or additions. In an aspect, a variant is a “functionalvariant” which retains some or all of the ability of the referencepolypeptide.

The term “functional variant” further includes conservativelysubstituted variants. The term “conservatively substituted variant”refers to a peptide having an amino acid sequence that differs from areference peptide by one or more conservative amino acid substitutions,and maintains some or all of the activity of the reference peptide. A“conservative amino acid substitution” is a substitution of an aminoacid residue with a functionally similar residue. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another; the substitution of one charged or polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between threonine and serine; the substitutionof one basic residue such as lysine or arginine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another; or the substitution of one aromatic residue, such asphenylalanine, tyrosine, or tryptophan for another. Such substitutionsare expected to have little or no effect on the apparent molecularweight or isoelectric point of the protein or polypeptide. The phrase“conservatively substituted variant” also includes peptides wherein aresidue is replaced with a chemically-derivatized residue, provided thatthe resulting peptide maintains some or all of the activity of thereference peptide as described herein.

The term “variant,” in connection with the polypeptides of the subjecttechnology, further includes a functionally active polypeptide having anamino acid sequence at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, and even 100% identical to the amino acid sequence ofa reference polypeptide.

The term “homologous” in all its grammatical forms and spellingvariations refers to the relationship between polynucleotides orpolypeptides that possess a “common evolutionary origin,” includingpolynucleotides or polypeptides from superfamilies and homologouspolynucleotides or proteins from different species (Reeck et al., Cell50:667, 1987). Such polynucleotides or polypeptides have sequencehomology, as reflected by their sequence similarity, whether in terms ofpercent identity or the presence of specific amino acids or motifs atconserved positions. For example, two homologous polypeptides can haveamino acid sequences that are at least 75%, at least 76%, at least 77%,at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, and even 100% identical.

“Percent (%) amino acid sequence identity” with respect to the variantpolypeptide sequences of the subject technology refers to the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues of a reference polypeptide (such as, forexample, SEQ ID NO:6), after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity.

Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared. For example, the %amino acid sequence identity may be determined using the sequencecomparison program NCBI-BLAST2. The NCBI-BLAST2 sequence comparisonprogram may be downloaded from ncbi.nlm.nih.gov. NCBI BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask yes, strand=all,expected occurrences 10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62. In situations whereNCBI-BLAST2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y where Xis the number of amino acid residues scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Aand B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

In this sense, techniques for determining amino acid sequence“similarity” are well known in the art. In general, “similarity” refersto the exact amino acid to amino acid comparison of two or morepolypeptides at the appropriate place, where amino acids are identicalor possess similar chemical and/or physical properties such as charge orhydrophobicity. A so-termed “percent similarity” may then be determinedbetween the compared polypeptide sequences. Techniques for determiningnucleic acid and amino acid sequence identity also are well known in theart and include determining the nucleotide sequence of the mRNA for thatgene (usually via a cDNA intermediate) and determining the amino acidsequence encoded therein, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity”, as can two or more amino acid sequences. The programsavailable in the Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.), for example,the GAP program, are capable of calculating both the identity betweentwo polynucleotides and the identity and similarity between twopolypeptide sequences, respectively. Other programs for calculatingidentity or similarity between sequences are known by those skilled inthe art.

An amino acid position “corresponding to” a reference position refers toa position that aligns with a reference sequence, as identified byaligning the amino acid sequences. Such alignments can be done by handor by using well-known sequence alignment programs such as ClustalW2,Blast 2, etc.

Unless specified otherwise, the percent identity of two polypeptide orpolynucleotide sequences refers to the percentage of identical aminoacid residues or nucleotides across the entire length of the shorter ofthe two sequences.

“Coding sequence” is used according to its ordinary and customarymeaning as understood by a person of ordinary skill in the art, and isused without limitation to refer to a DNA sequence that encodes for aspecific amino acid sequence.

“Suitable regulatory sequences” is used according to its ordinary andcustomary meaning as understood by a person of ordinary skill in theart, and is used without limitation to refer to nucleotide sequenceslocated upstream (5′ non-coding sequences), within, or downstream (3′non-coding sequences) of a coding sequence, and which influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences may include promoters,translation leader sequences, introns, and polyadenylation recognitionsequences.

“Promoter” is used according to its ordinary and customary meaning asunderstood by a person of ordinary skill in the art, and is used withoutlimitation to refer to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent cell types, or at different stages of development, or inresponse to different environmental conditions. Promoters, which cause agene to be expressed in most cell types at most times, are commonlyreferred to as “constitutive promoters.” It is further recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, DNA fragments of different lengths may haveidentical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein, is used according to its ordinaryand customary meaning as understood by a person of ordinary skill in theart, and is used without limitation to refer to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the subject technology. “Over-expression”refers to the production of a gene product in transgenic or recombinantorganisms that exceeds levels of production in normal or non-transformedorganisms.

“Transformation” is used according to its ordinary and customary meaningas understood by a person of ordinary skill in the art, and is usedwithout limitation to refer to the transfer of a polynucleotide into atarget cell. The transferred polynucleotide can be incorporated into thegenome or chromosomal DNA of a target cell, resulting in geneticallystable inheritance, or it can replicate independent of the hostchromosomal. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “transformed,” “transgenic,” and “recombinant,” when usedherein in connection with host cells, are used according to theirordinary and customary meanings as understood by a person of ordinaryskill in the art, and are used without limitation to refer to a cell ofa host organism, such as a plant or microbial cell, into which aheterologous nucleic acid molecule has been introduced. The nucleic acidmolecule can be stably integrated into the genome of the host cell, orthe nucleic acid molecule can be present as an extrachromosomalmolecule. Such an extrachromosomal molecule can be auto-replicating.Transformed cells, tissues, or subjects are understood to encompass notonly the end product of a transformation process, but also transgenicprogeny thereof.

The terms “recombinant,” “heterologous,” and “exogenous,” when usedherein in connection with polynucleotides, are used according to theirordinary and customary meanings as understood by a person of ordinaryskill in the art, and are used without limitation to refer to apolynucleotide (e.g., a DNA sequence or a gene) that originates from asource foreign to the particular host cell or, if from the same source,is modified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell buthas been modified through, for example, the use of site-directedmutagenesis or other recombinant techniques. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a position orform within the host cell in which the element is not ordinarily found.

Similarly, the terms “recombinant,” “heterologous,” and “exogenous,”when used herein in connection with a polypeptide or amino acidsequence, means a polypeptide or amino acid sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, recombinant DNAsegments can be expressed in a host cell to produce a recombinantpolypeptide.

The terms “plasmid,” “vector,” and “cassette” are used according totheir ordinary and customary meanings as understood by a person ofordinary skill in the art, and are used without limitation to refer toan extra chromosomal element often carrying genes which are not part ofthe central metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described, for example, by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual, 2^(nd) ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., In Current Protocols in Molecular Biology, published by GreenePublishing and Wiley-Interscience, 1987; the entireties of each of whichare hereby incorporated herein by reference to the extent they areconsistent herewith.

As used herein, “synthetic” or “organically synthesized” or “chemicallysynthesized” or “organically synthesizing” or “chemically synthesizing”or “organic synthesis” or “chemical synthesis” are used to refer topreparing the compounds through a series of chemical reactions; thisdoes not include extracting the compound, for example, from a naturalsource.

The term “orally consumable product” as used herein refers to anybeverage, food product, dietary supplement, nutraceutical,pharmaceutical composition, dental hygienic composition and cosmeticproduct which are contacted with the mouth of man or animal, includingsubstances that are taken into and subsequently ejected from the mouthand substances which are drunk, eaten, swallowed, or otherwise ingested;and that are safe for human or animal consumption when used in agenerally acceptable range of concentrations.

The term “food product” as used herein refers to fruits, vegetables,juices, meat products such as ham, bacon and sausage; egg products,fruit concentrates, gelatins and gelatin-like products such as jams,jellies, preserves, and the like; milk products such as ice cream, sourcream, yogurt, and sherbet; icings, syrups including molasses; corn,wheat, rye, soybean, oat, rice and barley products, cereal products, nutmeats and nut products, cakes, cookies, confectionaries such as candies,gums, fruit flavored drops, and chocolates, chewing gum, mints, creams,icing, ice cream, pies and breads. “Food product” also refers tocondiments such as herbs, spices and seasonings, flavor enhancers, suchas monosodium glutamate. “Food product” further refers to also includesprepared packaged products, such as dietetic sweeteners, liquidsweeteners, tabletop flavorings, granulated flavor mixes which uponreconstitution with water provide non-carbonated drinks, instant puddingmixes, instant coffee and tea, coffee whiteners, malted milk mixes, petfoods, livestock feed, tobacco, and materials for baking applications,such as powdered baking mixes for the preparation of breads, cookies,cakes, pancakes, donuts and the like. “Food product” also refers to dietor low-calorie food and beverages containing little or no sucrose.

As used herein, the term “stereoisomer” is a general term for allisomers of individual molecules that differ only in the orientation oftheir atoms in space. “Stereoisomer” includes enantiomers and isomers ofcompounds with more than one chiral center that are not mirror images ofone another (diastereomers).

As used herein, the term “amorphous rebaudioside D2” refers to anon-crystalline solid form of rebaudioside D2.

As used herein, the term “sweetness intensity” refers to the relativestrength of sweet sensation as observed or experienced by an individual,e.g., a human, or a degree or amount of sweetness detected by a taster,for example on a Brix scale.

As used herein, the term “enhancing the sweetness” refers to the effectof rebaudioside D2 in increasing, augmenting, intensifying,accentuating, magnifying, and/or potentiating the sensory perception ofone or more sweetness characteristics of a beverage product or aconsumable product of the present disclosure without changing the natureor quality thereof, as compared to a corresponding orally consumableproduct that does not contain rebaudioside D2.

As used herein, the term “off-taste(s)” refers to an amount or degree oftaste that is not characteristically or usually found in a beverageproduct or a consumable product of the present disclosure. For example,an off-taste is an undesirable taste of a sweetened consumable toconsumers, such as, a bitter taste, a licorice-like taste, a metallictaste, an aversive taste, an astringent taste, a delayed sweetnessonset, a lingering sweet aftertaste, and the like, etc.

As used herein, the term “w/v-%” refers to the weight of a compound,such as a sugar, (in grams) for every 100 ml of a liquid orallyconsumable product of the present disclosure containing such compound.As used herein, the term “w/w-%” refers to the weight of a compound,such as a sugar, (in grams) for every gram of an orally consumableproduct of the present disclosure containing such compound.

As used herein, the term “ppm” refers to part(s) per million by weight,for example, the weight of a compound, such as rebaudioside D2 (inmilligrams) per kilogram of an orally consumable product of the presentdisclosure containing such compound (i.e., mg/kg) or the weight of acompound, such as rebaudioside D2 (in milligrams) per liter of an orallyconsumable product of the present disclosure containing such compound(i.e., mg/L); or by volume, for example the volume of a compound, suchas rebaudioside D2 (in milliliters) per liter of an orally consumableproduct of the present disclosure containing such compound (i.e., ml/L).

In accordance with the present disclosure, a non-caloric sweetener andmethods for synthesizing the non-caloric sweetener are disclosed. Alsoin accordance with the present disclosure an enzyme and methods of usingthe enzyme to prepare the non-caloric sweetener are disclosed.

Synthetic Non-Caloric Sweetener: Synthetic Rebaudioside D2

In one aspect, the present disclosure is directed to a syntheticnon-caloric sweetener. The synthetic non-caloric sweetener is asynthetic rebaudioside-type steviol glycoside and has been given thename, “Rebaudioside D2”. As illustrated in the chemical structure (I)below, rebaudioside D2 (“Reb D2”) is a steviol glycoside having fiveglycosidic residues similar to the five glycosidic residues of thesteviol glycoside, rebaudioside D.

Rebaudioside D2 has the molecular formula C₅₀H₈₀O₂₈ and the IUPAC name,13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-6-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

As illustrated in the chemical structures for rebaudioside D2,rebaudioside E and rebaudioside D, rebaudioside D2 and rebaudioside Dhave five β-D-glucosyl units connected to the aglycone steviol in thestructure, whereas rebaudioside E contains four D-glycosidic residues(see e.g., Table 1 and FIG. 8). The synthesized rebaudioside D2 includestwo glycosidic residues at the C19 position and three glycosidicresidues at the C13 position of steviol. In comparison, rebaudioside Dalso includes five glycosidic residues; two glycosidic residues at theC19 position and three glycosidic residues at the C13 position of theaglycone steviol. The fifth glycosidic residue (“sugar V”) ofrebaudioside D2 is positioned at the C-6′ of the C13 O-glucose of Reb Eby a 1,6 β glycosidic linkage, whereas the fifth glycosidic residue(“sugar V”) of rebaudioside D is positioned at the C-3′ of the C13O-glucose of Reb E by a 1,3 β glycosidic linkage (see, FIG. 8).Rebaudioside E, however, includes two glycosidic residues at the C19position and two glycosidic residues at the C13 position. Without beingbound by theory, it is believed that steviol glycosides having 5glycosidic residues (rebaudioside D) and 4 glycosidic residues(rebaudioside A and rebaudioside E) have significantly better tastequality than steviol glycosides having less glycosidic residues(stevioside and rubusoside).

Methods of Producing Rebaudioside D2

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D2 from rebaudioside E. In one embodiment, themethod includes preparing a reaction mixture including rebaudioside E; asubstrate selected from the group consisting of sucrose, uridinediphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and aUDP-glycosyltransferase selected from a uridine diphosphoglycosyltransferase and a UDP-glycosyltransferase fusion enzyme (EUS)comprising a uridine-diphospho (UDP) glycosyltransferase domain coupledto a sucrose synthase domain; and incubating the reaction mixture for asufficient time to produce rebaudioside D2, wherein a glucose iscovalently coupled to the rebaudioside E to produce rebaudioside D2.

A particularly suitable a uridine diphospho (UDP) glycosyltransferasecan be, for example, EUGT11 (as described in WO 2013022989). EUGT11 is auridine 5′-diphosphate-dependent glycosyl transferase (“UGT”) having1,2-19-O-glucose and 1,2-13-O-glucose glycosylation activity. EUGT11 isknown to catalyze the production of stevioside to rebaudioside E andrebaudioside A to rebaudioside D. Surprisingly and unexpectedly,however, it has been discovered that uridine diphospho (UDP)glycosyltransferase can be used in vitro to convert rebaudioside E intorebaudioside D2.

A suitable uridine diphospho glycosyltransferase can be, for example, anOryza sativa uridine diphospho glycosyltransferase EUGT11. Aparticularly suitable uridine diphospho glycosyltransferase has theamino acid sequence of SEQ ID NO:1.

The method can further include adding a sucrose synthase to the reactionmixture that contains the uridine diphospho (UDP) glycosyltransferase.Addition of the sucrose synthase to the reaction mixture that includes auridine diphospho glycosyltransferase creates a “UGT-SUS couplingsystem”. In the UGT-SUS coupling system, UDP-glucose can be regeneratedfrom UDP and sucrose, which allows for omitting the addition of extraUDP-glucose to the reaction mixture or using UDP in the reactionmixture.

Suitable sucrose synthase domains can be for example, an Arabidopsissucrose synthase 1; a Coffea sucrose synthase 1 and a Stevia sucrosesynthase 1. A particularly suitable sucrose synthase domain can be, forexample, Arabidopsis sucrose synthase 1. A particularly suitableArabidopsis sucrose synthase 1 is Arabidopsis thaliana sucrose synthase1 (AtSUS1). A particularly suitable sucrose synthase 1 can be, forexample, a sucrose synthase 1 having the amino acid sequence of SEQ IDNO:3.

In another embodiment, the UDP-glycosyltransferase can be aUDP-glycosyltransferase fusion enzyme (also referred to herein as “EUS”)that includes a uridine diphospho glycosyltransferase domain coupled toa sucrose synthase domain. The UDP-glycosyltransferase fusion enzyme isdescribed in more detail below.

In the reaction, the UDP-glycosyltransferase (for example, EUGT11 andEUS) has a 1,6-13 O glucose glycosylation activity and, in oneembodiment, can transfer a glucose molecule to rebaudioside E to formrebaudioside D2. The UDP-glycosyltransferase (for example, EUGT11 andEUS) also has 1,2-19 O-glucose and 1,2-13-O-glucose glycosylationactivity. In another embodiment, the UDP-glycosyltransferase cantransfer a glucose molecule to stevioside to form rebaudioside E and canalso transfer a glucose molecule to rebaudioside A to form rebaudiosideD. Additionally, the EUS fusion enzyme has sucrose synthase activity,and thus, can regenerate UDP-glucose from UDP and sucrose.

A particularly suitable embodiment is directed to a method of producingrebaudioside D2 from rebaudioside E using a UGT-SUS coupling system. Themethod includes preparing a reaction mixture including rebaudioside E;sucrose; uridine diphosphate (UDP); a uridine diphosphoglycosyltransferase; and a sucrose synthase; and incubating the reactionmixture for a sufficient time to produce rebaudioside D2, wherein aglucose is covalently coupled to the rebaudioside E to producerebaudioside D2.

Suitable UDP glycosyltransferases for use in the method of thisembodiment is the same as described above. Suitable sucrose synthasesfor use in the method of this embodiment is the same as described above.

Another particularly suitable embodiment is directed to a method ofproducing rebaudioside D2 from rebaudioside E using aUDP-glycosyltransferase fusion enzyme that includes a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain. Themethod includes preparing a reaction mixture including rebaudioside E;sucrose; uridine diphosphate (UDP); and a UDP-glycosyltransferase fusionenzyme that includes a uridine diphospho glycosyltransferase domaincoupled to a sucrose synthase domain; and incubating the reactionmixture for a sufficient time to produce rebaudioside D2, wherein aglucose is covalently coupled to the rebaudioside E to producerebaudioside D2.

A particularly suitable UDP-glycosyltransferase fusion enzyme isdescribed in more detail below.

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D2 from stevioside. The method forsynthesizing rebaudioside D2 from stevioside includes preparing areaction mixture including stevioside; a substrate selected from thegroup consisting of sucrose, uridine diphosphate (UDP) and uridinediphosphate-glucose (UDP-glucose); and a UDP-glycosyltransferaseselected from the group consisting of a uridine diphosphoglycosyltransferase and a UDP-glycosyltransferase fusion enzyme thatincludes a uridine diphospho glycosyltransferase domain coupled to asucrose synthase domain; and incubating the reaction mixture for asufficient time to produce rebaudioside D2, wherein a glucose iscovalently coupled to the stevioside to produce a rebaudioside Eintermediate, and wherein a glucose is covalently coupled to therebaudioside E intermediate to produce rebaudioside D2.

Initially, the UDP glycosyltransferase for use in the method of thisembodiment is the same as described above. As described above, themethod may further include adding a sucrose synthase to the reactionmixture that contains the uridine diphospho glycosyltransferase tocreate a UGT-SUS coupling system.

A particularly suitable embodiment is directed to a method of producingrebaudioside D2 from stevioside using a UGT-SUS coupling system. Themethod includes preparing a reaction mixture including stevioside;sucrose; uridine diphosphate (UDP); a uridine diphosphoglycosyltransferase; and a sucrose synthase; and incubating the reactionmixture for a sufficient time to produce rebaudioside D2, wherein aglucose is covalently coupled to the stevioside to produce arebaudioside E intermediate, and wherein a glucose is covalently coupledto the rebaudioside E intermediate to produce rebaudioside D2.

Suitable UDP glycosyltransferases for use in the method of thisembodiment is the same as described above. Suitable sucrose synthasesfor use in the method of this embodiment is the same as described above.

Another particularly suitable embodiment is directed to a method ofproducing rebaudioside D2 from stevioside using aUDP-glycosyltransferase fusion enzyme that includes a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain. Themethod includes preparing a reaction mixture including stevioside; asubstrate selected from the group consisting of sucrose; uridinediphosphate (UDP); and a UDP-glycosyltransferase fusion enzyme thatincludes a uridine diphospho glycosyltransferase domain coupled to asucrose synthase domain; and incubating the reaction mixture for asufficient time to produce rebaudioside D2, wherein a glucose iscovalently coupled to the stevioside to produce a rebaudioside Eintermediate, and wherein a glucose is covalently coupled to therebaudioside E intermediate to produce rebaudioside D2.

A particularly suitable UDP-glycosyltransferase fusion enzyme isdescribed in more detail below.

UDP-Glycosyltransferase Fusion Enzyme

In another aspect, the present disclosure is directed to aUDP-glycosyltransferase fusion enzyme (also referred to herein as“EUS”). In particular, the UDP-glycosyltransferase fusion enzymeincludes a uridine diphospho glycosyltransferase domain coupled to asucrose synthase domain. The EUS fusion enzyme has a 1,2-19 O-glucoseglycosylation activity. Surprisingly and unexpectedly, the EUS fusionenzyme also has a 1,6-13 O-glucose glycosylation activity that cantransfer a glucose molecule to rebaudioside E to form rebaudioside D2.Additionally, the EUS fusion enzyme has sucrose synthase activity, andthus, can regenerate UDP-glucose from UDP and sucrose.

The UDP-glycosyltransferase fusion enzyme can have a polypeptidesequence with at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% and even 100%identical to the amino acid sequence set forth in SEQ ID NO:5. Suitably,the amino acid sequence of the UDP-glycosyltransferase fusion enzyme hasat least 80% identity to SEQ ID No:5. More suitably, the amino acidsequence of the UDP-glycosyltransferase fusion enzyme has at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, andeven 100% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:5.

In another aspect, the present disclosure relates to an isolated nucleicacid having a nucleotide sequence encoding the UDP-glycosyltransferasefusion enzyme described herein. For example, the isolated nucleic acidcan include a nucleotide sequence encoding a polypeptide of theUDP-glycosyltransferase fusion enzyme having a nucleic acid sequencethat has at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, andeven 100% sequence homology to the nucleic acid sequence set forth inSEQ ID NO:6. Suitably, the isolated nucleic acid includes a nucleotidesequence encoding a polypeptide of the UDP-glycosyltransferase fusionenzyme having an amino acid sequence that is at least 80% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:5. Moresuitably, the isolated nucleic acid includes a nucleotide sequenceencoding a polypeptide of the UDP-glycosyltransferase fusion enzymehaving an amino acid sequence that has at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, and even 100%sequence identity to the amino acid sequence set forth in SEQ ID NO:5.The isolated nucleic acid thus includes those nucleotide sequencesencoding functional fragments of SEQ ID NO:5, functional variants of SEQID NO:5, or other homologous polypeptides that have, for example, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, and even 100% sequence identity to SEQ ID NO:5.As known by those skilled in the art, the nucleic acid sequence encodingthe UDP-glycosyltransferase can be codon optimized for expression in asuitable host organism such as, for example, bacteria and yeast.

A suitable uridine diphospho glycosyltransferase domain can be an Oryzasativa uridine diphospho glycosyltransferase EUGT11 (GenBank AccessionNo. AC133334). A particularly suitable uridine diphosphoglycosyltransferase EUGT11 can be, for example, the uridine diphosphoglycosyltransferase EUGT11 domain having an amino acid sequence of SEQID NO:1.

Suitable sucrose synthase domains can be for example, an Arabidopsissucrose synthase 1; a Coffea sucrose synthase 1 and a Stevia sucrosesynthase 1. A particularly suitable sucrose synthase domain can be, forexample, Arabidopsis sucrose synthase 1. A particularly suitableArabidopsis sucrose synthase 1 is Arabidopsis thaliana sucrose synthase1 (AtSUS1). A particularly suitable sucrose synthase 1 domain can be,for example, a sucrose synthase 1 having the amino acid sequence of SEQID NO:3.

Sucrose synthase catalyzes the chemical reaction between NDP-glucose andD-fructose to produce NDP and sucrose. Sucrose synthase is aglycosyltransferase. The systematic name of this enzyme class isNDP-glucose:D-fructose 2-alpha-D-glucosyltransferase. Other names incommon use include UDP glucose-fructose glucosyltransferase, sucrosesynthetase, sucrose-UDP glucosyltransferase, sucrose-uridine diphosphateglucosyltransferase, and uridine diphosphoglucose-fructoseglucosyltransferase.

Methods of Producing Rebaudioside D

In another aspect, the present disclosure is directed to a method forsynthesizing rebaudioside D. The method includes preparing a reactionmixture including rebaudioside A; a substrate selected from the groupconsisting of sucrose, uridine diphosphate (UDP) and uridinediphosphate-glucose (UDP-glucose); and a UDP-glycosyltransferaseselected from the group consisting of uridine diphosphoglycosyltransferase and a UDP-glycosyltransferase fusion enzyme (EUS)that includes a uridine diphospho glycosyltransferase domain coupled toa sucrose synthase domain; and incubating the reaction mixture for asufficient time to produce rebaudioside D, wherein a glucose iscovalently coupled to the rebaudioside A to produce rebaudioside D.

In the embodiment wherein the UDP-glycosyltransferase is uridinediphospho glycosyltransferase, a suitable uridine diphosphoglycosyltransferase can be an Oryza sativa uridine diphosphoglycosyltransferase EUGT11 (GenBank Accession No. AC133334). Aparticularly suitable uridine diphospho glycosyltransferase can be, forexample, the uridine diphospho glycosyltransferase having an amino acidsequence of SEQ ID NO:1.

In the embodiment wherein the UDP-glycosyltransferase is a uridinediphospho glycosyltransferase, the method can further include adding asucrose synthase to the reaction mixture. Suitable sucrose synthases canbe for example, an Arabidopsis sucrose synthase 1; a Coffea sucrosesynthase 1 and a Stevia sucrose synthase 1. A particularly suitablesucrose synthase can be, for example, Arabidopsis sucrose synthase 1. Aparticularly suitable Arabidopsis sucrose synthase 1 is Arabidopsisthaliana sucrose synthase 1 (AtSUS1). A particularly suitable sucrosesynthase 1 can be, for example, a sucrose synthase 1 having the aminoacid sequence of SEQ ID NO:3.

In the embodiment wherein the UDP-glycosyltransferase is aUDP-glycosyltransferase fusion enzyme (EUS) that includes a uridinediphospho glycosyltransferase domain coupled to a sucrose synthasedomain as described above, a suitable uridine diphosphoglycosyltransferase domain can be an Oryza sativa uridine diphosphoglycosyltransferase EUGT11 (GenBank Accession No. AC133334). Aparticularly suitable uridine diphospho glycosyltransferase domain canbe, for example, a uridine diphospho glycosyltransferase domain havingan amino acid sequence of SEQ ID NO:1. A particularly suitable sucrosesynthase 1 domain can have, for example, an amino acid sequence of SEQID NO:3. A particularly suitable UDP-glycosyltransferase fusion enzyme(EUS) can have, for example, an amino acid sequence of SEQ ID NO:5.

A particularly suitable embodiment is directed to a method forsynthesizing rebaudioside D using a UGT-SUS coupling system. The methodincludes preparing a reaction mixture including rebaudioside A; sucrose;uridine diphosphate (UDP); a UDP-glycosyltransferase; and a sucrosesynthase; and incubating the reaction mixture for a sufficient time toproduce rebaudioside D, wherein a glucose is covalently coupled to therebaudioside A to produce rebaudioside D.

Suitable UDP glycosyltransferases for use in the method of thisembodiment is the same as described above. Suitable sucrose synthasesfor use in the method of this embodiment is the same as described above.

A particularly suitable embodiment is directed to a method forsynthesizing rebaudioside D using a UDP-glycosyltransferase fusionenzyme. The method includes preparing a reaction mixture includingrebaudioside A; sucrose; uridine diphosphate (UDP); and aUDP-glycosyltransferase fusion enzyme (EUS) that includes a uridinediphospho glycosyltransferase domain coupled to a sucrose synthasedomain; and incubating the reaction mixture for a sufficient time toproduce rebaudioside D, wherein a glucose is covalently coupled to therebaudioside A to produce rebaudioside D.

A particularly suitable UDP-glycosyltransferase fusion enzyme isdescribed in more detail above.

Orally Consumable Products

In another aspect, the present disclosure is directed to an orallyconsumable product having a sweetening amount of rebaudioside D2,selected from the group consisting of a beverage product and aconsumable product.

The orally consumable product can have a sweetness intensity equivalentto about 1% (w/v-%) to about 4% (w/v-%) sucrose solution.

The orally consumable product can have from about 5 ppm to about 100 ppmrebaudioside D2.

The rebaudioside D2 can be the only sweetener in the orally consumableproduct.

The orally consumable product can also have at least one additionalsweetener. The at least one additional sweetener can be a natural highintensity sweetener, for example. The additional sweetener can beselected from a stevia extract, a steviol glycoside, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F, dulcoside A, rubusoside, steviolbioside,sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose,rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK,aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone(NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, mogrosideIV, siamenoside I, mogroside V, monatin, thaumatin, monellin, brazzein,L-alanine, glycine, Lo Han Guo, hernandulcin, phyllodulcin, trilobtain,and combinations thereof.

The orally consumable product can also have at least one additive. Theadditive can be, for example, a carbohydrate, a polyol, an amino acid orsalt thereof, a polyamino acid or salt thereof, a sugar acid or saltthereof, a nucleotide, an organic acid, an inorganic acid, an organicsalt, an organic acid salt, an organic base salt, an inorganic salt, abitter compound, a flavorant, a flavoring ingredient, an astringentcompound, a protein, a protein hydrolysate, a surfactant, an emulsifier,a flavonoids, an alcohol, a polymer, and combinations thereof.

In one aspect, the present disclosure is directed to a beverage productcomprising a sweetening amount of rebaudioside D2.

The beverage product can be, for example, a carbonated beverage productand a non-carbonated beverage product. The beverage product can also be,for example, a soft drink, a fountain beverage, a frozen beverage; aready-to-drink beverage; a frozen and ready-to-drink beverage, coffee,tea, a dairy beverage, a powdered soft drink, a liquid concentrate,flavored water, enhanced water, fruit juice, a fruit juice flavoreddrink, a sport drink, and an energy drink.

In some embodiments, a beverage product of the present disclosure caninclude one or more beverage ingredients such as, for example,acidulants, fruit juices and/or vegetable juices, pulp, etc.,flavorings, coloring, preservatives, vitamins, minerals, electrolytes,erythritol, tagatose, glycerine, and carbon dioxide. Such beverageproducts may be provided in any suitable form, such as a beverageconcentrate and a carbonated, ready-to-drink beverage.

In certain embodiments, beverage products of the present disclosure canhave any of numerous different specific formulations or constitutions.The formulation of a beverage product of the present disclosure can varyto a certain extent, depending upon such factors as the product'sintended market segment, its desired nutritional characteristics, flavorprofile, and the like. For example, in certain embodiments, it cangenerally be an option to add further ingredients to the formulation ofa particular beverage product. For example, additional (i.e., moreand/or other) sweeteners can be added, flavorings, electrolytes,vitamins, fruit juices or other fruit products, tastents, masking agentsand the like, flavor enhancers, and/or carbonation typically may beadded to any such formulations to vary the taste, mouthfeel, nutritionalcharacteristics, etc. In embodiments, the beverage product can be a colabeverage that contains water, about 5 ppm to about 100 ppm rebaudiosideD2, an acidulant, and flavoring. Exemplary flavorings can be, forexample, cola flavoring, citrus flavoring, and spice flavorings. In someembodiments, carbonation in the form of carbon dioxide can be added foreffervescence. In other embodiments, preservatives can be added,depending upon the other ingredients, production technique, desiredshelf life, etc. In certain embodiments, caffeine can be added. In someembodiments, the beverage product can be a cola-flavored carbonatedbeverage, characteristically containing carbonated water, sweetener,kola nut extract and/or other flavoring, caramel coloring, one or moreacids, and optionally other ingredients.

Suitable amounts of rebaudioside D2 present in the beverage product canbe, for example, from about 5 ppm to about 100 ppm. In some embodiments,low concentrations of rebaudioside D2, for example, less than 100 ppm,and has an equivalent sweetness to sucrose solutions havingconcentrations between 10,000 ppm to 30,000 ppm. The final concentrationthat ranges from about 5 ppm to about 100 ppm, from about 5 ppm to about95 ppm, from about 5 ppm to about 90 ppm, from about 5 ppm to about 85ppm, from about 5 ppm to about 80 ppm, from about 5 ppm to about 75 ppm,from about 5 ppm to about 70 ppm, from about 5 ppm to about 65 ppm, fromabout 5 ppm to about 60 ppm, from about 5 ppm to about 55 ppm, fromabout 5 ppm to about 50 ppm, from about 5 ppm to about 45 ppm, fromabout 5 ppm to about 40 ppm, from about 5 ppm to about 35 ppm, fromabout 5 ppm to about 30 ppm, from about 5 ppm to about 25 ppm, fromabout 5 ppm to about 20 ppm, from about 5 ppm to about 15 ppm, or fromabout 5 ppm to about 10 ppm. Alternatively, rebaudioside D2 can bepresent in beverage products of the present disclosure at a finalconcentration that ranges from about 5 ppm to about 100 ppm, from about10 ppm to about 100 ppm, from about 15 ppm to about 100 ppm, from about20 ppm to about 100 ppm, from about 25 ppm to about 100 ppm, from about30 ppm to about 100 ppm, from about 35 ppm to about 100 ppm, from about40 ppm to about 100 ppm, from about 45 ppm to about 100 ppm, from about50 ppm to about 100 ppm, from about 55 ppm to about 100 ppm, from about60 ppm to about 100 ppm, from about 65 ppm to about 100 ppm, from about70 ppm to about 100 ppm, from about 75 ppm to about 100 ppm, from about80 ppm to about 100 ppm, from about 85 ppm to about 100 ppm, from about90 ppm to about 100 ppm, or from about 95 ppm to about 100 ppm.

In another aspect, the present disclosure is directed to a consumablecomprising a sweetening amount of rebaudioside D2. The consumable canbe, for example, a food product, a nutraceutical, a pharmaceutical, adietary supplement, a dental hygienic composition, an edible gelcomposition, a cosmetic product and a tabletop flavoring.

As used herein, “dietary supplement(s)” refers to compounds intended tosupplement the diet and provide nutrients, such as vitamins, minerals,fiber, fatty acids, amino acids, etc. that may be missing or may not beconsumed in sufficient quantities in a diet. Any suitable dietarysupplement known in the art may be used. Examples of suitable dietarysupplements can be, for example, nutrients, vitamins, minerals, fiber,fatty acids, herbs, botanicals, amino acids, and metabolites.

As used herein, “nutraceutical(s)” refers to compounds, which includesany food or part of a food that may provide medicinal or healthbenefits, including the prevention and/or treatment of disease ordisorder (e.g., fatigue, insomnia, effects of aging, memory loss, mooddisorders, cardiovascular disease and high levels of cholesterol in theblood, diabetes, osteoporosis, inflammation, autoimmune disorders,etc.). Any suitable nutraceutical known in the art may be used. In someembodiments, nutraceuticals can be used as supplements to food andbeverages and as pharmaceutical formulations for enteral or parenteralapplications which may be solid formulations, such as capsules ortablets, or liquid formulations, such as solutions or suspensions.

In some embodiments, dietary supplements and nutraceuticals can furthercontain protective hydrocolloids (such as gums, proteins, modifiedstarches), binders, film-forming agents, encapsulating agents/materials,wall/shell materials, matrix compounds, coatings, emulsifiers, surfaceactive agents, solubilizing agents (oils, fats, waxes, lecithins, etc.),adsorbents, carriers, fillers, co-compounds, dispersing agents, wettingagents, processing aids (solvents), flowing agents, taste-maskingagents, weighting agents, jellyfying agents, gel-forming agents,antioxidants and antimicrobials.

As used herein, a “gel” refers to a colloidal system in which a networkof particles spans the volume of a liquid medium. Although gels mainlyare composed of liquids, and thus exhibit densities similar to liquids,gels have the structural coherence of solids due to the network ofparticles that spans the liquid medium. For this reason, gels generallyappear to be solid, jelly-like materials. Gels can be used in a numberof applications. For example, gels can be used in foods, paints, andadhesives. Gels that can be eaten are referred to as “edible gelcompositions.” Edible gel compositions typically are eaten as snacks, asdesserts, as a part of staple foods, or along with staple foods.Examples of suitable edible gel compositions can be, for example, geldesserts, puddings, jams, jellies, pastes, trifles, aspics,marshmallows, gummy candies, and the like. In some embodiments, ediblegel mixes generally are powdered or granular solids to which a fluid maybe added to form an edible gel composition. Examples of suitable fluidscan be, for example, water, dairy fluids, dairy analogue fluids, juices,alcohol, alcoholic beverages, and combinations thereof. Examples ofsuitable dairy fluids can be, for example, milk, cultured milk, cream,fluid whey, and mixtures thereof. Examples of suitable dairy analoguefluids can be, for example, soy milk and non-dairy coffee whitener.

As used herein, the term “gelling ingredient” refers to any materialthat can form a colloidal system within a liquid medium. Examples ofsuitable gelling ingredients can be, for example, gelatin, alginate,carageenan, gum, pectin, konjac, agar, food acid, rennet, starch, starchderivatives, and combinations thereof. It is well known to those in theart that the amount of gelling ingredient used in an edible gel mix oran edible gel composition can vary considerably depending on a number offactors such as, for example, the particular gelling ingredient used,the particular fluid base used, and the desired properties of the gel.

Gel mixes and gel compositions of the present disclosure can be preparedby any suitable method known in the art. In some embodiments, edible gelmixes and edible gel compositions of the present disclosure can beprepared using other ingredients in addition to rebaudioside D2 and thegelling agent. Examples of other suitable ingredients can be, forexample, a food acid, a salt of a food acid, a buffering system, abulking agent, a sequestrant, a cross-linking agent, one or moreflavors, one or more colors, and combinations thereof.

Any suitable pharmaceutical composition known in the art may be used. Incertain embodiments, a pharmaceutical composition of the presentdisclosure can contain from about 5 ppm to about 100 ppm of rebaudiosideD2, and one or more pharmaceutically acceptable excipients. In someembodiments, pharmaceutical compositions of the present disclosure canbe used to formulate pharmaceutical drugs containing one or more activeagents that exert a biological effect. Accordingly, in some embodiments,pharmaceutical compositions of the present disclosure can contain one ormore active agents that exert a biological effect. Suitable activeagents are well known in the art (e.g., The Physician's Desk Reference).Such compositions can be prepared according to procedures well known inthe art, for example, as described in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., USA.

Rebaudioside D2 can be used with any suitable dental and oral hygienecompositions known in the art. Examples of suitable dental and oralhygiene compositions can be, for example, toothpastes, tooth polishes,dental floss, mouthwashes, mouthrinses, dentrifices, mouth sprays, mouthrefreshers, plaque rinses, dental pain relievers, and the like.

Suitable amounts of rebaudioside D2 present in the consumable can be,for example, from about 5 parts per million (ppm) to about 100 parts permillion (ppm). In some embodiments, low concentrations of rebaudiosideD2, for example, less than 100 ppm, has an equivalent sweetness tosucrose solutions having concentrations between 10,000 ppm to 30,000ppm. The final concentration that ranges from about 5 ppm to about 100ppm, from about 5 ppm to about 95 ppm, from about 5 ppm to about 90 ppm,from about 5 ppm to about 85 ppm, from about 5 ppm to about 80 ppm, fromabout 5 ppm to about 75 ppm, from about 5 ppm to about 70 ppm, fromabout 5 ppm to about 65 ppm, from about 5 ppm to about 60 ppm, fromabout 5 ppm to about 55 ppm, from about 5 ppm to about 50 ppm, fromabout 5 ppm to about 45 ppm, from about 5 ppm to about 40 ppm, fromabout 5 ppm to about 35 ppm, from about 5 ppm to about 30 ppm, fromabout 5 ppm to about 25 ppm, from about 5 ppm to about 20 ppm, fromabout 5 ppm to about 15 ppm, or from about 5 ppm to about 10 ppm.Alternatively, rebaudioside D2 can be present in consumable products ofthe present disclosure at a final concentration that ranges from about 5ppm to about 100 ppm, from about 10 ppm to about 100 ppm, from about 15ppm to about 100 ppm, from about 20 ppm to about 100 ppm, from about 25ppm to about 100 ppm, from about 30 ppm to about 100 ppm, from about 35ppm to about 100 ppm, from about 40 ppm to about 100 ppm, from about 45ppm to about 100 ppm, from about 50 ppm to about 100 ppm, from about 55ppm to about 100 ppm, from about 60 ppm to about 100 ppm, from about 65ppm to about 100 ppm, from about 70 ppm to about 100 ppm, from about 75ppm to about 100 ppm, from about 80 ppm to about 100 ppm, from about 85ppm to about 100 ppm, from about 90 ppm to about 100 ppm, or from about95 ppm to about 100 ppm.

In certain embodiments, from about 5 ppm to about 100 ppm ofrebaudioside D2 is present in food product compositions. As used herein,“food product composition(s)” refers to any solid or liquid ingestiblematerial that can, but need not, have a nutritional value and beintended for consumption by humans and animals.

Examples of suitable food product compositions can be, for example,confectionary compositions, such as candies, mints, fruit flavoreddrops, cocoa products, chocolates, and the like; condiments, such asketchup, mustard, mayonnaise, and the like; chewing gums; cerealcompositions; baked goods, such as breads, cakes, pies, cookies, and thelike; dairy products, such as milk, cheese, cream, ice cream, sourcream, yogurt, sherbet, and the like; tabletop sweetener compositions;soups; stews; convenience foods; meats, such as ham, bacon, sausages,jerky, and the like; gelatins and gelatin-like products such as jams,jellies, preserves, and the like; fruits; vegetables; egg products;icings; syrups including molasses; snacks; nut meats and nut products;and animal feed.

Food product compositions can also be herbs, spices and seasonings,natural and synthetic flavors, and flavor enhancers, such as monosodiumglutamate. In some embodiments, food product compositions can be, forexample, prepared packaged products, such as dietetic sweeteners, liquidsweeteners, granulated flavor mixes, pet foods, livestock feed, tobacco,and materials for baking applications, such as powdered baking mixes forthe preparation of breads, cookies, cakes, pancakes, donuts and thelike. In other embodiments, food product compositions can also be dietand low-calorie food and beverages containing little or no sucrose.

In certain embodiments that may be combined with any of the precedingembodiments, the rebaudioside D2 is the only sweetener, and the producthas a sweetness intensity equivalent to about 1% to about 4% (w/v-%)sucrose solution. In certain embodiments that can be combined with anyof the preceding embodiments, the consumable products and beverageproducts can further include an additional sweetener, where the producthas a sweetness intensity equivalent to about 1% to about 10% (w/v-%)sucrose solution. In certain embodiments that can be combined with anyof the preceding embodiments, every sweetening ingredient in the productis a high intensity sweetener. In certain embodiments that can becombined with any of the preceding embodiments, every sweeteningingredient in the product can a natural high intensity sweetener. Incertain embodiments that can be combined with any of the precedingembodiments, the additional sweetener contains one or more sweetenersselected from a stevia extract, a steviol glycoside, stevioside,rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside F, dulcoside A, rubusoside, steviolbioside, sucrose, highfructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose,erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame,neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC),neohesperidin dihydrochalcone (NDHC), rubusoside mogroside IV,siamenoside I, mogroside V, monatin, thaumatin, monellin, brazzein,L-alanine, glycine, Lo Han Guo, hernandulcin, phyllodulcin, trilobtain,and combinations thereof. In certain embodiments that can be combinedwith any of the preceding embodiments, the consumable products andbeverage products can further include one or more additives selectedfrom a carbohydrate, a polyol, an amino acid or salt thereof, apoly-amino acid or salt thereof, a sugar acid or salt thereof, anucleotide, an organic acid, an inorganic acid, an organic salt, anorganic acid salt, an organic base salt, an inorganic salt, a bittercompound, a flavorant, a flavoring ingredient, an astringent compound, aprotein, a protein hydrolysate, a surfactant, an emulsifier, aflavonoids, an alcohol, a polymer, and combinations thereof. In certainembodiments that can be combined with any of the preceding embodiments,the rebaudioside D2 has a purity of about 50% to about 100% by weightbefore it is added into the product.

Sweetener

In another aspect, the present disclosure is directed to a sweetenerconsisting of a chemical structure:

The sweetener can further include at least one of a filler, a bulkingagent and an anticaking agent. Suitable fillers, bulking agents andanticaking agents are known in the art.

In certain embodiments, rebaudioside D2 sweetener can be included and/oradded at a final concentration that is sufficient to sweeten and/orenhance the sweetness of the consumable products and beverage products.The “final concentration” of rebaudioside D2 refers to the concentrationof rebaudioside D2 present in the final consumable products and beverageproducts (i.e., after all ingredients and/or compounds have been addedto produce the consumable products and beverage products). Accordingly,in certain embodiments, rebaudioside D2 is included and/or added to acompound or ingredient used to prepare the consumable products andbeverage products. The rebaudioside D2 may be present in a singlecompound or ingredient, or multiple compounds and ingredients. In otherembodiments, rebaudioside D2 is included and/or added to the consumableproducts and beverage products. In certain preferred embodiments, therebaudioside D2 is included and/or added at a final concentration thatranges from about 5 ppm to about 100 ppm, from about 5 ppm to about 95ppm, from about 5 ppm to about 90 ppm, from about 5 ppm to about 85 ppm,from about 5 ppm to about 80 ppm, from about 5 ppm to about 75 ppm, fromabout 5 ppm to about 70 ppm, from about 5 ppm to about 65 ppm, fromabout 5 ppm to about 60 ppm, from about 5 ppm to about 55 ppm, fromabout 5 ppm to about 50 ppm, from about 5 ppm to about 45 ppm, fromabout 5 ppm to about 40 ppm, from about 5 ppm to about 35 ppm, fromabout 5 ppm to about 30 ppm, from about 5 ppm to about 25 ppm, fromabout 5 ppm to about 20 ppm, from about 5 ppm to about 15 ppm, or fromabout 5 ppm to about 10 ppm. Alternatively, the rebaudioside D2 isincluded and/or added at a final concentration that ranges from about 5ppm to about 100 ppm, from about 10 ppm to about 100 ppm, from about 15ppm to about 100 ppm, from about 20 ppm to about 100 ppm, from about 25ppm to about 100 ppm, from about 30 ppm to about 100 ppm, from about 35ppm to about 100 ppm, from about 40 ppm to about 100 ppm, from about 45ppm to about 100 ppm, from about 50 ppm to about 100 ppm, from about 55ppm to about 100 ppm, from about 60 ppm to about 100 ppm, from about 65ppm to about 100 ppm, from about 70 ppm to about 100 ppm, from about 75ppm to about 100 ppm, from about 80 ppm to about 100 ppm, from about 85ppm to about 100 ppm, from about 90 ppm to about 100 ppm, or from about95 ppm to about 100 ppm. For example, rebaudioside D2 may be includedand/or added at a final concentration of about 5 ppm, about 10 ppm,about 15 ppm, about 20 ppm, about 25 ppm, about 30 ppm, about 35 ppm,about 40 ppm, about 45 ppm, about 50 ppm, about 55 ppm, about 60 ppm,about 65 ppm, about 70 ppm, about 75 ppm, about 80 ppm, about 85 ppm,about 90 ppm, about 95 ppm, or about 100 ppm, including any range inbetween these values.

In certain embodiments, rebaudioside D2 is the only sweetener includedand/or added to the consumable products and the beverage products. Insuch embodiments, the consumable products and the beverage products havea sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrosesolution, about 1% to about 3% (w/v-%) sucrose solution, or about 1% toabout 2% (w/v-%) sucrose solution. Alternatively, the consumableproducts and the beverage products have a sweetness intensity equivalentto about 1% to about 4% (w/v-%) sucrose solution, about 2% to about 4%(w/v-%) sucrose solution, about 3% to about 4% (w/v-%) sucrose solution,or about 4%. For example, the consumable products and the beverageproducts may have a sweetness intensity equivalent to about 1%, about2%, about 3%, or about 4% (w/v-%) sucrose solution, including any rangein between these values.

The consumable products and beverage products of the present disclosurecan include a mixture of rebaudioside D2 and one or more sweeteners ofthe present disclosure in a ratio sufficient to achieve a desirablesweetness intensity, nutritional characteristic, taste profile,mouthfeel, or other organoleptic factor.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1

In this Example, full-length DNA fragments of all candidate UGT geneswere synthesized.

Specifically, the cDNAs were codon optimized for E. coli expression(Genscript, Piscataway, N.J.). The synthesized DNA was cloned into abacterial expression vector pETite N-His SUMO Kan Vector (Lucigen). Forthe nucleotide sequence encoding the UDP-glycosyltransferase fusionenzyme (EUS) (see, SEQ ID NO:6), a GSG-linker (encoded by the nucleotidesequence: ggttctggt) was inserted in frame between a nucleotide sequenceencoding the Oryza sativa uridine diphospho glycosyltransferase (EUGT11)domain (see, SEQ ID NO:2) and the nucleotide sequence encoding the A.thaliana sucrose synthase 1 (AtSUS1) domain (see, SEQ ID NO:4). Table 2summarizes the protein and sequence identifier numbers.

TABLE 2 Sequence Identification Numbers. Name SEQ ID NO DescriptionEUGT11 SEQ ID NO: 1 Amino acid EUGT11 SEQ ID NO: 2 Nucleic acid AtSUS1SEQ ID NO: 3 Amino acid AtSUS1 SEQ ID NO: 4 Nucleic acid EUS fusionenzyme SEQ ID NO: 5 Amino acid EUS fusion enzyme SEQ ID NO: 6 Nucleicacid

Each expression construct was transformed into E. coli BL21 (DE3), whichwas subsequently grown in LB media containing 50 μg/mL kanamycin at 37°C. until reaching an OD₆₀₀ of 0.8-1.0. Protein expression was induced byaddition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and theculture was further grown at 16° C. for 22 hr. Cells were harvested bycentrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collectedand were either used immediately or stored at −80° C.

The cell pellets were re-suspended in lysis buffer (50 mM potassiumphosphate buffer, pH 7.2, 25 μg/ml lysozyme, 5 μg/ml DNase I, 20 mMimidazole, 500 mM NaCl, 10% glycerol, and 0.4% TRITON X-100). The cellswere disrupted by sonication at 4° C., and the cell debris was clarifiedby centrifugation (18,000×g; 30 min). The supernatant was loaded to aequilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen)affinity column. After loading of protein sample, the column was washedwith equilibration buffer to remove unbound contaminant proteins. TheHis-tagged UGT recombinant polypeptides were eluted by equilibrationbuffer containing 250 mM imidazole. After purification, the recombinantEUGT11 protein (62 kD band indicated by arrow in FIG. 2A) and the EUSfusion enzyme (155 kD band indicated by arrow in FIG. 2B) were analyzedby SDS-PAGE. Molecular weight standards are indicated to left of eachSDS-gel image.

Example 2

In this Example, recombinant EUGT11 protein and recombinant EUS fusionenzyme were assayed for 1,2-19 O-glucose glycosylation activity usingrebaudioside A as the steviol glycoside substrate.

The recombinant polypeptides (10 μg) were tested in a 200 μL in vitroreaction system. The reaction system contained 50 mM potassium phosphatebuffer, pH 7.2, 3 mM MgCl₂, 1 mg/ml steviol glycoside substrate, and 1mM UDP-glucose. The reaction was performed at 30° C. and terminated byadding 200 μL of 1-butanol. The samples were extracted three times with200 μL of 1-butanol. The pooled fraction was dried and dissolved in 70μL of 80% methanol for high-performance liquid chromatography (HPLC)analysis. Rebaudioside A (purity 99%) was used as the substrate.Rebaudioside A was obtained from Blue California (Rancho SantaMargarita, Calif.). In vitro reactions were carried out for 14 hours and24 hours. FIG. 3A shows the peak for rebaudioside D (labeled “Reb D”)for comparison.

The UGT catalyzed glycosylation reaction was coupled to a UDP-glucosegenerating reaction (referred to herein as the “UGT-SUS couplingsystem”) catalyzed by a sucrose synthase (e.g., AtSUS1). Specifically,the Arabidopsis thaliana sucrose synthase 1 (AtSUS1) sequence(Bieniawska et al., Plant J. 2007, 49: 810-828) was synthesized andinserted into a bacterial expression vector. The recombinant AtSUS1protein was expressed and purified by affinity chromatography. TheAtSUS1 protein was added to the EUGT11 protein to form an in vitroreaction mixture referred to herein as the EUGT11-AtSUS1 couplingsystem. In the resultant UGT-SUS (e.g., EUGT11-AtSUS1) coupling system,the UDP-glucose was generated from sucrose and UDP, such that theaddition of an extra UDP-glucose was omitted.

HPLC analysis was performed using a Dionex UPLC ultimate 3000 system(Sunnyvale, Calif.), including a quaternary pump, a temperaturecontrolled column compartment, an auto sampler and a UV absorbancedetector. A Phenomenex Luna NH2 with guard column, 150×3.0 mm, 3 μm (100A) was used for the characterization of steviol glycosides. 72%acetonitrile in water was used for isocratic elution in HPLC analysis.

As shown in FIG. 3, EUS and EUGT11 transferred a sugar moiety torebaudioside A to produce rebaudioside D in all reaction conditions.Rebaudioside A was completely converted to rebaudioside D by the EUSfusion enzyme (FIGS. 3B and 3E) and the UGT-SUS (i.e., EUGT11-AtSUS1)coupling reaction system (FIGS. 5D and 5G) at incubation times of 14hours and 24 hours. However, rebaudioside A was only partially convertedto rebaudioside D at 14 hours (FIG. 3C) and 24 hours (FIG. 3F) by theEUGT11 enzyme alone. Furthermore, the same molecule amount of EUS had ahigher enzymatic activity than the EUGT11 and converted all of therebaudioside A to rebaudioside D at incubation times of 14 hours (FIG.3B) and 24 hours (FIG. 3E). These results demonstrated that the reactionof the UGT-SUS (i.e., EUGT11-AtSUS1) coupling system could be achievedusing the EUS fusion enzyme. Additionally, these results demonstratedthat EUGT11 showed a 1,2-19 O-glucose glycosylation activity to producerebaudioside D from rebaudioside A (FIGS. 3C and 3F) and that AtSUS1enhanced the conversion efficiency by EUGT11 in the UGT-SUS couplingsystem (FIGS. 3B, 3D, 3E and 3G). FIG. 3A shows peaks for stevioside(labeled “Ste”), rebaudioside A (labeled “Reb A”) and rebaudioside D(labeled “Reb D”) for comparison.

Example 3

In this Example, EUGT11 and EUS were assayed for 1,2-19 O-glucoseglycosylation activity using stevioside as the steviol glycosidesubstrate at incubation times of 14 hours and 24 hours as described inExample 2.

In addition to the conversion of rebaudioside A to rebaudioside D byEUGT11 as discussed in Example 2 above, EUGT11 also converted steviosideto rebaudioside E (labeled “Reb E” in FIG. 4). Surprisingly, anunexpected compound, rebaudioside D2 (labelled “Reb D2” in FIG. 4),having a HPLC retention time of about 7.28 minutes was produced by bothEUGT11 and EUS in all reactions. When AtSUS1 was added to the EUGT11reaction mixture to create the UGT-SUS coupling system (FIGS. 4D and 4G)and when EUS was used (FIGS. 4B and 4E), more rebaudioside D2 wasproduced. Along with the increase in rebaudioside D2 production,rebaudioside E (labeled “Reb E” in FIGS. 4C and 4F) that was producedwas consumed during the production of the rebaudioside D2. These resultsindicated that EUGT11 can catalyze the reaction to produce arebaudioside (rebaudioside D2) from rebaudioside E. FIG. 4A shows peaksfor stevioside (labeled “Ste”), rebaudioside A (labeled “Reb A”) andrebaudioside D (labeled “Reb D”) for comparison.

Example 4

In this Example, to confirm the conversion of rebaudioside E torebaudioside D2 in vitro, EUGT11 and EUS were assayed using rebaudiosideE as the steviol glycoside substrate as described in Example 2.

For comparison, the enzymatic activities of another UGT (UGT76G1) wasalso assayed. UGT76G1, from stevia, has been identified as an enzymethat transfers a sugar residue to C-3′ of the C13 O-glucose ofstevioside to form rebaudioside A. As shown in FIGS. 5F and 5J, whenUGT76G1 was used in the UGT-SUS coupling system, a sugar residue wastransferred to the C-3′ of the C13 O-glucose of rebaudioside E to formrebaudioside D. FIGS. 5A and 5B show purified rebaudioside D (“Reb D”)and rebaudioside E (“Reb-E”) for comparison.

As discussed in Example 3 above and shown in FIGS. 5C and 5G, EUGT11alone could transfer one glucose molecule to rebaudioside E to form arebaudioside (referred to herein as “rebaudioside D2” and labeled “RebD2” in FIGS. 5C and 5G) that was distinct from rebaudiosides D and E(compare peaks in FIG. 5A and FIG. 5B, respectively). EUGT11 in theUGT-SUS coupling system (FIGS. 5D and 5H) and EUS (FIGS. 5E and 5I)enhanced the conversion from rebaudioside E to rebaudioside D2.

These results demonstrated that EUGT11 is a UGT with 1,2-19 O-glucoseglycosylation activity to produce related steviol glycosides. EUGT11 cancatalyze the reaction to produce Reb-E from stevioside as substrate andReb D from Reb A as substrate. Surprisingly, a compound (Reb D2) wasunexpectedly synthesized in the in vitro reaction with stevioside assubstrate. Further experiments confirmed that Reb D2 was directlysynthesized from Reb E. According to the structure of Reb D2, in the invitro reaction, EUGT11 transferred a D-glucose to the C-6′ of the C13O-glucose of Reb-E to generate a 1,6-β-glycosidic linkage.

Example 5

In this Example, the rebaudioside (rebaudioside D2) was purified from anenlarged in vitro reaction and prepared for liquid chromatography/massspectrometry (LC/MS) and nuclear magnetic resonance (NMR) analysis.

The rebaudioside (rebaudioside D2) was purified from an enlarged invitro reaction. The in vitro reaction was monitored by HPLC analysis. Atthe desired time point, Reb D2 compound was purified by column andconcentrated by vacuum drying. The purified Reb D2 was white powder with95% purity. The collected Reb D2 compound was used for High ResolutionMass Spectra (HRMS) analysis. HRMS data were generated with a LTQOrbitrap Discovery HRMS instrument, with its resolution set to 30 k anddata was scanned from m/z 150 to 1500 in positive ion electrospray mode.The needle voltage was set to 4 kV; the other source conditions weresheath gas=25, aux gas=0, sweep gas=5 (all gas flows in arbitraryunits), capillary voltage=30V, capillary temperature=300° C., and tubelens voltage=75. The sample was diluted with 2:2:1acetonitrile:methanol:water (same as infusion eluent) and 50 microlitersof sample were injected. Nuclear Magnetic Resonance (NMR) spectra wereacquired using a Bruker Avance DRX 500 MHz instrument or a Varian INOVA600 MHz instrument using standard pulse sequences. The 1D (¹H and ¹³C)and 2D (TOCSY, HMQC, and HMBC) NMR spectra were performed in pyridine-d5(also known as C₅D₅N).

The molecular formula of Reb D2 was deduced as C₅₀H₈₀O₂₈ on the basis ofits positive high resolution mass spectrum (HRMS) which showed adductions corresponding to [M+NH₄]⁺ and [M+Na]⁺ at m/z 1146.5169 and1151.4721 respectively; this composition was supported by the ¹³C NMRspectral data. The ¹H NMR spectral data of Reb D2 showed the presence oftwo methyl singlets at δ 1.10 and 1.44, two olefinic protons as singletsat δ 5.09 and 5.72 of an exocyclic double bond, nine sp3 methylene andtwo sp3 methine protons between δ 0.74-2.80, which is characteristic forthe ent-kaurane diterpenoids isolated from the genus Stevia.

The basic skeleton of ent-kaurane diterpenoids was supported by theTOCSY studies which showed key correlations: H-1/H-2; H-2/H-3; H-5/H-6;H-6/H-7; H-9/H-11; H-11/H-12. The ¹H NMR spectrum of Reb D2 also showedthe presence of anomeric protons resonating at δ 5.04, 5.10, 5.21, 5.48,and 6.30; suggesting five sugar units in its structure. Acid hydrolysisof Reb D2 with 5% H₂SO₄ afforded D-glucose which was identified bydirect comparison with an authentic sample by TLC. The large couplingconstants observed for the five anomeric protons of the glucose moietiesat δ 5.04 (d, J=7.5 Hz), 5.10 (d, J=7.4 Hz), 5.21 (d, J=7.9 Hz), 5.48(d, J=7.9 Hz), and 6.30 (d, J=7.9 Hz), suggested their β-orientation asreported for other steviol glycosides. The ¹H and ¹³C NMR values for RebD2 were assigned on the basis of TOCSY, HMQC and HMBC data and aresummarized in Table 3.

TABLE 3 ¹H and ¹³C NMR values for Reb D2 and Reb E. Reb D2 (1) Reb E(2)Position ¹H NMR ¹³C NMR ¹H NMR ¹³C NMR  1 0.74 t 41.2 0.73 t 41 (12.8),(13.3), 1.67 m 1.68 m  2 1.48 m, 20.6 1.46 m, 20.6 2.12 m 2.13 m  3 1.13m, 38.4 1.12 m, 38.2 2.80 d 2.78 d (12.8) (12.8)  4 — 44.9 — 44.8  50.98 d 58.1 0.97 d 38.2 (11.8) (11.8)  6 1.87 m, 22.6 1.85 m, 22.6 2.10m 2.09 m  7 1.28 m, 42.2 1.27 m, 42.1 1.64 m 1.63 m  8 — 43.3 — 43  90.88 d, 54.5 0.88 br s 54.5 (7.5) 10 — 40.3 — 40.2 11 1.66 m 21.2 1.65 m21.1 12 1.91 m, 38.2 1.96 m, 37.8 2.22 m 2.16 m 13 — 86.8 — 86.6 14 1.69d, 44.9 1.74 d, 44.8 (11.4), (11.4), 2.49 d, 2.54 d, (11.0) (11.0) 152.04 m, 48.6 2.04 m, 48.5 2.16 m 2.12 m 16 — 155 — 154.9 17 5.09 s,105.4 5.09 s, 105.4 5.72 s 5.76 s 18 1.44 s 29.9 1.43 s 29.8 19 — 176.3— 176.2 20 1.10 s 17.3 1.10 s 17.2  1′ 6.30 d 93.9 6.30 d 93.9 (7.9)(7.9)  2′ 4.38 m 81.5 4.38 m 81.7  3′ 4.27 m 78.5 4.26 m 78.4  4′ 4.24 m72 4.22 m 72.1  5′ 3.94 m 79.6 3.92 m 79.5  6′ 4.33 m, 62.7 4.33 m, 62.64.43 m 4.43 m  1″ 5.10 d 98.3 5.16 d 98.4 (7.4) (7.5)  2″ 4.18 m 84.84.17 m 84.9  3″ 4.29 m 78.6 4.32 m 78.5  4″ 4.20 m 71.3 4.22 m 71.8  5″3.78 m 78.5 3.72 m 78.2  6″ 4.32 m, 69.8 4.26 m, 62.9 4.57 m 4.35 m  1′″5.21 d 107.2 5.32 d 107.2 (7.9) (7.5)  2′″ 4.14 t 77.6 4.15 t 77.7 (8.4)(8.4)  3′″ 4.25 m 78.7 4.26 m 78.6  4′″ 4.34 m 72.4 4.36 m 72.3  5′″3.94 m 79.1 3.96 m 79  6′″ 4.43 m, 63.3 4.46 m, 63.2 4.53 m 4.56 m  1″″5.48 d 106.2 5.48 d 106.2 (7.9) (7.9)  2″″ 4.04 t 76.8 4.06 t 76.8 (7.9)(7.9)  3″″ 4.22 m 78.8 4.25 m 78.7  4″″ 4.32 m 71.2 4.31 m 71.2  5″″3.99 m 79.1 4.02 m 79.1  6″″ 4.38 m, 63.5 4.42 m, 63.4 4.55 m 4.54 m 1″″ 5.04 d 105.9 (7.5)  2″″′ 4.02 m 77  3″″′ 4.21 m 78.6  4″″′ 4.25 m72.2  5″″′ 3.96 m 79.1  6″″′ 4.34 m, 63.3 4.48 m

Based on the extensive 1D (¹H and ¹³C), 2D NMR (TOCSY, HMQC, and HMBC)and high resolution mass spectral (HRMS) data, the structure of Reb D2was identified and compared to the structure of rebaudioside E (see,FIG. 6).

Based on the results from NMR spectral data and hydrolysis experimentsof Reb D2, it was concluded that there were five β-D-glucosyl units inits structure connected to the aglycone steviol. A close comparison ofthe ¹H and ¹³C NMR values of Reb D2 with rebaudioside E (see, Table 2)suggested the presence of a steviol aglycone moiety with a2-O-β-D-glucobiosyl unit at C13 in the form of an ether linkage andanother 2-O-β-D-glucobiosyl unit at C19 position in the form of an esterlinkage, leaving the assignment of the additional β-D-glucosyl unit.Further, from the ¹³C NMR spectral data of Reb D2 which showed that oneof the five oxymethine carbons of sugar moieties appeared downfield at δ69.8, suggested the placement of the additional β-D-glucosyl unit atthis position. Identical proton and carbon spectral data for the twosugars I and IV in Reb D2 and Reb E suggested the placement of theadditional β-D-glucosyl unit at the 6-position of either sugar II orsugar III. The downfield shift for both the ¹H and ¹³C chemical shiftsat the 6-position of sugar II of the β-D-glucosyl moiety suggested theadditional β-D-glucosyl unit was attached at this position. Thestructure was further supported by the key TOCSY and HMBC correlationsas shown in FIG. 7.

Based on the results of NMR and mass spectral data as well as hydrolysisstudies, the structure of Reb D2 produced by the enzymatic conversion ofrebaudioside E was deduced as13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester.

13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester (Reb D2). White powder; ¹H-NMR (600 MHz, C₅D₅N, δ ppm) and ¹³C-NMR(150 MHz, C₅D₅N, δ ppm) spectroscopic data see Table 2; HRMS (M+NH₄)⁺m/z 1146.5169 (calcd. for C₅₀H₈₄O₂₈N: 1146.5180), (M+Na)⁺ m/z 1151.4721(calcd. for C₅₀H₈₀O₂₈Na: 1151.4734).

To a solution of Reb D2 (5 mg) in MeOH (10 ml) was added 3 ml of 5%H₂SO₄ and the mixture was refluxed for 24 hours. The reaction mixturewas then neutralized with saturated sodium carbonate and extracted withethyl acetate (EtOAc) (2×25 ml) to give an aqueous fraction containingsugars and an EtOAc fraction containing the aglycone part. The aqueousphase was concentrated and compared with standard sugars using the TLCsystems EtOAc/n-butanol/water (2:7:1) and CH₂Cl₂/MeOH/water (10:6:1);the sugar was identified as D-glucose.

The structure of Reb D2 was confirmed as13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester on the basis of extensive 1D (¹H and ¹³C), and 2D NMR (TOCSY,HMQC, and HMBC), as well as high resolution mass spectral data andhydrolysis studies.

Example 6

In this Example, the structure of the rebaudioside (rebaudioside D2) wascompared to Reb-E and Reb-D.

According to the steviol glycosides database, only Reb-D contains fiveβ-D-glucosyl units in its structure connected to the aglycone steviol,two glycosidic residues at the C19 position and three glycosidicresidues at the C13 position of the aglycone steviol. UGT76G1 fromstevia has been identified as an enzyme that transfers a sugar residueto C-3′ of the C13 O-glucose of Reb E to form rebaudioside D (see, FIGS.5F and 5J). Reb D2 is a steviol glycoside that also contains fiveD-glycosidic residues with different structure comparison torebaudioside D (see, FIG. 8). The fifth glycosidic residue (“sugar V”)of rebaudioside D2 is positioned at the C-6′ of the C-13-O glucose ofReb E by a 1,6 β glycosidic linkage, whereas the fifth glycosidicresidue (“sugar V”) of rebaudioside D is positioned at the C-3′ of theC13 O-glucose of Reb E by a 1,3 β glycosidic linkage (see, FIG. 8). Asdescribed herein, both EUGT11 and EUG can directly convert Reb E intoReb D2 in vitro.

Example 7

In this Example, a taste test was conducted on Reb D2.

Sensory evaluation of Reb D2 was performed using sucrose as a control.The sucrose sample purchased from Sigma-Aldrich and prepared controlsamples at three different concentrations of 1.0%, 3.0%, and 6.0%sucrose in bottled water (w/v) at room temperature. The steviolglycoside Reb D2 at 300, and 600 ppm for sensory evaluation was preparedby adding corresponding mass into a 1000 mL of bottled water. Themixture was stirred at room temperature and the steviol glycoside samplewas then evaluated against several control sucrose samples at 1.0%,3.0%, and 6.0% by a panel of nine volunteers.

The blind results showed consistent results among majority of ninevolunteers at two different concentrations (300 and 600 ppm) of the RebD2; the overall % sweetness equivalence (SE) averages were about 2.4 and5.4, respectively. The result indicates that the rebaudioside D2 isabout 80-90 times sweeter to sucrose.

Example 8

In this Example, the solubility of Reb D2 was compared to Reb D.

Reb D2 and Reb D were added to water to prepare solutions with 0.25 mM,0.5 mM, 1 mM, 1.5 mM, 2 mM, 5 mM and 10 mM Reb D2 and Reb D. Reb D2powder completely dissolved in water immediately, however only 0.25 mMReb D totally dissolved in water. Additionally, solutions of Reb D atconcentrations of 0.5 mM, 1 mM, 1.5 mM, 2 mM, 5 mM and 10 mM did notdissolve when heated at 30° C. for 72 hours.

These results demonstrate that Reb D2 has a higher solubility in waterthan does Reb D.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and systems withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

What is claimed is:
 1. A method for synthesizing rebaudioside D, themethod comprising: preparing a reaction mixture comprising rebaudiosideA; a substrate selected from the group consisting of sucrose, uridinediphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and aUDP-glycosyltransferase fusion enzyme comprising a uridine diphosphoglycosyltransferase domain coupled to a sucrose synthase domain; whereinthe uridine diphospho glycosyltransferase domain is an Oryza sativauridine diphospho glycosyltransferase EUGT11 comprising the amino acidsequence of SEQ ID NO: 1, and the sucrose synthase domain is selectedfrom the group consisting of an Arabidopsis sucrose synthase 1, a Coffeasucrose synthase 1, and a Stevia sucrose synthase 1, and incubating thereaction mixture for a sufficient time to produce rebaudioside D,wherein a glucose is covalently coupled to the rebaudioside A to producerebaudioside D.
 2. The method of claim 1, wherein theUDP-glycosyltransferase fusion enzyme comprises an amino acid sequencehaving about 90% sequence identity to SEQ ID NO:5.
 3. The method ofclaim 1, wherein the sucrose synthase domain is an Arabidopsis thalianasucrose synthase
 1. 4. The method of claim 1, wherein the sucrosesynthase domain comprises the amino acid sequence of SEQ ID NO:3.
 5. Themethod of claim 1, wherein the UDP-glycosyltransferase fusion enzyme isexpressed in a host organism.
 6. The method of claim 5, wherein the hostorganism is a bacterial cell.
 7. The method of claim 5, wherein the hostorganism is a yeast cell.
 8. The method of claim 5, wherein the hostorganism is an E. coli cell.
 9. The method of claim 2, wherein theUDP-glycosyltransferase fusion enzyme comprises an amino acid sequencehaving about 95% sequence identity to SEQ ID NO:
 5. 10. The method ofclaim 9, wherein the UDP-glycosyltransferase fusion enzyme comprises theamino acid sequence of SEQ ID NO: 5.